Apical constriction is an ancient developmental process that triggers key morphogenetic events such as neurulation in vertebrates and gastrulation in insects. However, the pathways involved are not fully understood. During pupariation, presumptive leg joints undergo apical constriction and epithelial invagination to initiate joint morphogenesis. We hypothesize that the process is mediated by an increase in actomyosin contractility and a decrease in cell-cell adhesion. The goal of this grant is to study the roles of the Rho GTPases Rho1, Rac1 and Cdc42 and their regulators, the RhoGEFs and RhoGAPs, in controlling the stability and turnover of adherence junctions (AJs), and the contribution of these genes to apical constriction and epithelial invagination during joint morphogenesis. We find that the inhibition of Rho1 or the activation of Rac1 or Cdc42 blocks joint morphogenesis. We also find that RhoGAP68F (GAP68F), a putative GAP for Cdc42, and RhoGAP5A (GAP5A), a putative GAP for Rac1, are expressed at presumptive joints and are required to initiate joint morphogenesis. We provide strong preliminary evidence to suggest that these regulators act through distinct pathways to control apical constriction. To test the role of the Rho GTPases and their regulators in apical constriction we propose the following specific AIMs: AIM1: We will test the hypothesis that Rho1, Cdc42 and Rac1 respectively regulate the influx, efflux and the stable pool of AJs at the ZA to promote apical constriction. AIM2: Large-scale interaction screens identified Rab4 and Sec3 as partners of GAP68F suggesting that GAP68F inhibits endocytic recycling from two distinct recycling routes to decrease the surface expression of AJs and thereby adhesive cell-cell contacts. In agreement, we find accumulation of GAP68F in the endocytic compartment. Therefore, we will test the hypothesis that GAP68F inhibits endocytic recycling and thereby adhesive cell-cell contacts to promote apical constriction. AIM3: We find that GAP5A is enriched at or near the zonula adherence (ZA). We will test the hypothesis that GAP5A acts at the ZA to decrease the stability of AJs and thereby adhesive cell-cell contacts to promote apical constriction. The successful completion of these studies will highlight the contribution of junctional stability and endocytic trafficking to apical constriction and epithelial invagination with implications to vertebrate systems.